Oligonucleotides are DNA, RNA or DNA/RNA hybrid molecules that in general are relatively short, synthetic, and single stranded. Synthetic oligonucleotides are inexpensive, readily available, and can be made for any desired sequence. These molecules are often labeled with molecular tracers to follow their presence in various assays. Such labeled molecules are commonly referred to as “probes”. The labeled oligonucleotide can be used as a hybridization probe to identify nucleic acids and studying the binding of proteins and other molecules to the probe. The results obtained using such assays depend in large part upon the specific activity of the probe. In general, probes of high specific activity that can be generated in a controllable and reproducible manner are most desirable.
Common nucleic acid labels currently include groups containing radioactive atoms such as 32P or 35S, biotin, fluorescent molecules (e.g. fluorescein and rhodamine derivatives) and enzymes whose activity is detectable, for example, as a result of catalyzing a reaction that produces a fluorescing or colorimetric reagent (e.g. horseradish peroxidase and alkaline phosphatase). In addition, nucleotide analogs conjugated to haptens (e.g. biotin, digoxigenin, etc.) may be used to label the nucleic acid. A hapten is subsequently recognized by a high affinity binding ligand (e.g., streptavidin) or an antibody, that is conjugated to a fluorescent marker or enzyme reagent for detection of the nucleic acid.
Generally, polynucleotide kinase (PNK) is used to radiolabel oligonucleotides with γ-32P-ATP, in particular at the 5′ phosphate. If there is no phosphate group present on the 5′ end of a nucleic acid molecule, PNK can add a radiolabeled phosphate to the free 5′hydroxyl group of the nucleic acid. Alternatively, if a 5′ phosphate is present, PNK can remove the non-radioactive 5′ phosphate and replace it with a radiolabeled phosphate in an exchange reaction. However, in either an addition or exchange reaction, the resulting labeled nucleic acid has only a single 32P label per nucleic acid molecule. No more than one 32P label can be introduced per nucleic acid molecule by PNK.
The use of PNK is also limited, moreover, because the enzyme is not completely efficient in the addition reaction and even less efficient in the exchange reaction. This low efficiency can result in variable incorporation of radiolabel and a diminished specific activity measured in counts per minute (cpm) per micromole of DNA or RNA substrate. Because of the inefficiency of the PNK, the enzyme reaction results leaves residual unlabeled nucleic acids. These unlabeled (“cold”) nucleic acids cannot be purified away from the radiolabeled (“hot”) nucleic acids because they have nearly identical chemical and physical properties. The labeled nucleic acid differs from the unlabeled molecule by the presence of only one additional phosphate group. The cold nucleic acid will then compete with the hot nucleic acid when the labeled nucleic acid is used in a practical application, e.g., binding to a target molecule, diminishing the probe's effectiveness.
In addition, because the unlabeled nucleic acid cannot be removed, the precise specific activity of the product generally cannot be determined. Usually, the quantity of oligonucleotide labeled is too small to measure the amount of product directly recovered from the reaction, e.g., by U.V. absorbence. Therefore, specific activity can only be estimated, either based on an assumption of the efficiency of recovery of the labeled nucleic acid or the recovery of unincorporated α-32P-ATP.
Nucleic acid molecules can also be labeled on their free 3′ hydroxyl group with a radiolabeled nucleotide, e.g., α-32P-dATP, using the enzyme Terminal Deoxynucleotidyl Transferase (TdT). As discussed below, TdT labeling, however, exhibits many of the same problems as PNK labeling. In particular, TdT does not efficiently label DNA, and, furthermore, the labeled nucleic acids cannot be separated from the unlabeled nucleic acids, reducing the efficiency of the method in which the labeled nucleic acid is being used.
Biologically, TdT is involved in increasing antigen receptor diversity by adding a variable number of nucleotides into chromosomal break points during the genetic rearrangement of the variable regions of immature T lymphocyte and B lymphocyte receptors (Gilfillan et al., 1995, 1 mm. Reviews 148:201). TdT adds an average of four nucleotides to chromosomal break points in vivo; however, the number of nucleotides added is extremely variable and highly dependent on local conditions (Rock et al., 1994, J. Exp. Med., 179:323). TdT also adds a variable number of nucleotides in vitro to the free 3′ end of a nucleic acid probe, which makes it difficult to accurately determine the specific activity of a probe or to produce the probe in a reproducible manner. Invariably, a mixture of species is formed from which it is very difficult to isolate a unique product. Thus, this method results in a heterogeneous reaction product ranging from unlabeled to hundred-fold labeled species. Further, TdT-labeled probes with a tail that is longer than 10 or 20 residues can produce high backgrounds when hybridized to complex nucleic acid samples as a result of binding to poly A tails of mRNAs or repeats in genomic DNA.
A number of schemes have been employed to increase the specific activity and sensitivity of oligonucleotide probes without compromising their favorable hybridization properties. Ullrich et al. (1984, Nature 309:418–425) and Studencki and Wallace (1984, DNA 3:7–15) described methods in which two overlapping sequence-specific oligonucleotides are labeled in a primer extension reaction to a specific activity that can exceed 10×109 CPM/μg. While effective, these methods result in a double stranded product, require two sequence-specific custom oligonucleotides, and use two to four radiolabeled substrate deoxynucleotide triphosphates, making such approaches more expensive and less convenient than labeling with PNK. Oligonucleotides having a dendrimeric structure at the 5′-end can be synthesized and labeled to high specific activity using PNK (Shchepinov et al., 1997, Nucleic Acids Res. 25:4447–4454). However these reagents are costly and are not readily available.
Both RNA and DNA polymerase-based techniques that incorporate labeled nucleotides into nucleic acids are well known in the art (see, e.g., Ausubel et al., 1992, “Short Protocols in Molecular Biology”, John Wiley & Sons, New York). For example, nick translation methods allow the preparation of uniformly labeled DNA probes for a variety of assays. Small breakages are introduced into a double stranded DNA of interest (for example by treating with DNase I), and the gaps are “filled in” using DNA polymerase in the presence of radiolabeled or non-isotopically labeled nucleotides. Alternatively, labeled probes are generated by random priming. Briefly, a double-stranded DNA of interest is denatured and the individual strands replicated in the presence of the Klenow fragment of E. coli DNA polymerase I and labeled nucleotides, using random hexamers as primers for the extension step. Alternatively, a DNA which is to be used as a probe can be generated by cutting a parent DNA with a restriction enzymes that generates a 5′ overhang, and the overhang filled in using the Klenow fragment of E. coli DNA polymerase I and labeled nucleotides. Yet another method of producing a labeled DNA probe is by generating the probe by polymerase chain reaction (PCR) in the presence of labeled nucleotides.
Generally, labeled RNA probes can be produced by carrying out the transcription reaction that generates the RNA in the presence of labeled nucleotides. Such transcription reactions most commonly utilize bacteriophage RNA polymerases, such as SP6, T3, and T7.
Another scheme for labeling oligonucleotides proposed by Bahl et al. (U.S. Pat. No. 5,538,872), involves a “bridging” technique that ligates a target molecule to a signal molecule via a “bridge” molecule. The signal molecule shows no complementarity towards the target molecule, and comprises a unique 4 or more nucleotide stretch at one end and is labeled at the other end. In addition to the sequence of interest, the target molecule comprises a unique 4 or more nucleotide stretch at the end opposite to that at which the unique sequence of the signal molecule is located. The signal and target molecules are ligated by a “bridge” molecule, consisting of two specific 4 or more nucleotide sequences, one complimentary to the unique nucleotide sequence in the target molecule, and the other complimentary to the unique nucleotide sequence in the signal molecule. The target molecule, the signal molecule and the bridging molecules are allowed to hybridize, allowing the target and signal molecules to align in a directional manner. The signal and target molecules are ligated together and the result is a double stranded oligonucleotide complex that can be left as is or disassociated into the bridge and a completed probe consisting of the target and signal molecules.
Another problem associated with conventional template-based labeling methods arises when the labeled probe must be separated from the template strand. Probe techniques have been attempted to resolve this problem. For example, Dattagupta et al., U.S. Pat. No. 4,808,520, proposed a probe that is labeled by hybridizing two oligonucleotides of equal length that have regions of mutual complementarity located at the 3′ ends of the oligonucleotides. One overhang is filled in with labeled nucleotides to generate a labeled probe using the other strand as a template. The template strand is not extended because it has a different nucleotide composition from the strand that is labeled, and only nucleotides required to generate the probe are added to the labeling reaction. The probe strand can then be separated from the template strand based on the differing sizes of the strands. The drawback to this method is that the template strand is designed in a sequence specific manner that depends on the oligonucleotide that is being labeled.
Clearly, therefore, a need exists for methods whereby highly purified labeled nucleic acids of high specific activity can be produced in a controllable, reproducible manner.